RÉSUMÉ
Resource partitioning is central to the incredible productivity of microbial communities, including gigatons in annual methane emissions through syntrophic interactions. Previous work revealed how a sulfate reducer (Desulfovibrio vulgaris, Dv) and a methanogen (Methanococcus maripaludis, Mm) underwent evolutionary diversification in a planktonic context, improving stability, cooperativity, and productivity within 300-1000 generations. Here, we show that mutations in just 15 Dv and 7 Mm genes within a minimal assemblage of this evolved community gave rise to co-existing ecotypes that were spatially enriched within a few days of culturing in a fluidized bed reactor. The spatially segregated communities partitioned resources in the simulated subsurface environment, with greater lactate utilization by attached Dv but partial utilization of resulting H2 by low affinity hydrogenases of Mm in the same phase. The unutilized H2 was scavenged by high affinity hydrogenases of planktonic Mm, producing copious amounts of methane. Our findings show how a few mutations can drive resource partitioning amongst niche-differentiated ecotypes, whose interplay synergistically improves productivity of the entire mutualistic community.
Sujet(s)
Desulfovibrio vulgaris , Écotype , Méthane , Methanococcus , Mutation , Méthane/métabolisme , Methanococcus/génétique , Methanococcus/métabolisme , Desulfovibrio vulgaris/génétique , Desulfovibrio vulgaris/métabolisme , Évolution moléculaire dirigée , Hydrogène/métabolisme , Microbiote/génétiqueRÉSUMÉ
Protein-protein interactions (PPIs) are some of the most challenging target classes in drug discovery. Highly sensitive detection techniques are required for the identification of chemical modulators of PPIs. Here, we introduce PPI confocal nanoscanning (PPI-CONA), a miniaturized, microbead based high-resolution fluorescence imaging assay. We demonstrate the capabilities of PPI-CONA by detecting low affinity ternary complex formation between the human CDC34A ubiquitin-conjugating (E2) enzyme, ubiquitin, and CC0651, a small molecule enhancer of the CDC34A-ubiquitin interaction. We further exemplify PPI-CONA with an E2 enzyme binding study on CC0651 and a CDC34A binding specificity study of a series of CC0651 analogues. Our results indicate that CC0651 is highly selective toward CDC34A. We further demonstrate how PPI-CONA can be applied to screening very low affinity interactions. PPI-CONA holds potential for high-throughput screening for modulators of PPI targets and characterization of their affinity, specificity, and selectivity.
RÉSUMÉ
Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. Various automated approaches have been proposed, many of which work well for high-contrast datasets where the features of interest can be easily detected and are clearly separated from one another. Our inner ear stereocilia cryo-electron tomographic datasets are characterized by a dense array of hexagonally packed actin filaments that are frequently cross-connected. These features make automated segmentation very challenging, further aggravated by the high-noise environment of cryo-electron tomograms and the high complexity of the densely packed features. Using prior knowledge about the actin bundle organization, we have placed layers of a highly simplified ball-and-stick actin model to first obtain a global fit to the density map, followed by regional and local adjustments of the model. We show that volumetric model building not only allows us to deal with the high complexity, but also provides precise measurements and statistics about the actin bundle. Volumetric models also serve as anchoring points for local segmentation, such as in the case of the actin-actin cross connectors. Volumetric model building, particularly when further augmented by computer-based automated fitting approaches, can be a powerful alternative when conventional automated segmentation approaches are not successful.
Sujet(s)
Actines , Cryomicroscopie électronique , Cryomicroscopie électronique/méthodes , Actines/composition chimique , Tomographie en microscopie électronique/méthodes , Animaux , Oreille interne/imagerie diagnostique , Cytosquelette d'actine/composition chimique , Cytosquelette d'actine/ultrastructureRÉSUMÉ
Antimicrobial resistance has emerged as a global public health threat, and development of novel therapeutics for treating infections caused by multi-drug resistant bacteria is urgent. Staphylococcus aureus is a major human and animal pathogen, responsible for high levels of morbidity and mortality worldwide. The intracellular survival of S. aureus in macrophages contributes to immune evasion, dissemination, and resilience to antibiotic treatment. Here, we present a confocal fluorescence imaging assay for monitoring macrophage infection by green fluorescent protein (GFP)-tagged S. aureus as a front-line tool to identify antibiotic leads. The assay was employed in combination with nanoscaled chemical analyses to facilitate the discovery of a new, active rifamycin analogue. Our findings indicate a promising new approach for the identification of antimicrobial compounds with macrophage intracellular activity. The antibiotic identified here may represent a useful addition to our armory in tackling the silent pandemic of antimicrobial resistance.
Sujet(s)
Rifamycine , Infections à staphylocoques , Animaux , Humains , Staphylococcus aureus , Protéines à fluorescence verte/génétique , Rifamycine/usage thérapeutique , Antibactériens/pharmacologie , Antibactériens/usage thérapeutique , Infections à staphylocoques/microbiologie , MacrophagesRÉSUMÉ
Lignocellulose biomass has a tremendous potential as renewable biomaterials for fostering the "bio-based society" and circular bioeconomy paradigm. It requires efficient use and breakdown of fiber cell walls containing mainly cellulose, hemicellulose and lignin biopolymers. Despite their great importance, there is an extensive debate on the true structure of fiber walls and knowledge on the macromolecular nano-organization is limited and remains elusive in 3D. We employed dual-axis electron tomography that allows visualization of previously unseen 3D macromolecular organization/biopolymeric nano-architecture of the secondary S2 layer of Norway spruce fiber wall. Unprecedented 3D nano-structural details with novel insights into cellulose microfibrils (~ 2 nm diameter), macrofibrils, nano-pore network and cell wall chemistry (volume %) across the S2 were explored and quantified including simulation of structure related permeability. Matrix polymer association with cellulose varied between microfibrils and macrofibrils with lignin directly associated with MFs. Simulated bio-nano-mechanical properties revealed stress distribution within the S2 and showed similar properties between the idealized 3D model and the native S2 (actual tomogram). Present work has great potential for significant advancements in lignocellulose research on nano-scale understanding of cell wall assembly/disassembly processes leading to more efficient industrial processes of functionalization, valorization and target modification technologies.
Sujet(s)
Lignine , Nanostructures , Lignine/métabolisme , Tomographie en microscopie électronique/méthodes , Cellulose/composition chimique , Paroi cellulaire/métabolismeRÉSUMÉ
Increased nuclear size correlates with lower survival rates and higher grades for prostate cancer. The short-chain dehydrogenase/reductase (SDR) family member DHRS7 was suggested as a biomarker for use in prostate cancer grading because it is largely lost in higher-grade tumors. Here, we found that reduction in DHRS7 from the LNCaP prostate cancer cell line with normally high levels of DHRS7 increases nuclear size, potentially explaining the nuclear size increase observed in higher-grade prostate tumors where it is lost. An exogenous expression of DHRS7 in the PC3 prostate cancer cell line with normally low DHRS7 levels correspondingly decreases nuclear size. We separately tested 80 compounds from the Microsource Spectrum library for their ability to restore normal smaller nuclear size to PC3 cells, finding that estradiol propionate had the same effect as the re-expression of DHRS7 in PC3 cells. However, the drug had no effect on LNCaP cells or PC3 cells re-expressing DHRS7. We speculate that separately reported beneficial effects of estrogens in androgen-independent prostate cancer may only occur with the loss of DHRS7/ increased nuclear size, and thus propose DHRS7 levels and nuclear size as potential biomarkers for the likely effectiveness of estrogen-based treatments.
Sujet(s)
Oestradiol , Tumeurs de la prostate , Mâle , Humains , Oestradiol/pharmacologie , Propionates , Tumeurs de la prostate/traitement médicamenteux , Prostate , Oestrogènes , OxidoreductasesRÉSUMÉ
A simplistic assumption in setting up a competition assay is that a low affinity labeled ligand can be more easily displaced from a target protein than a high affinity ligand, which in turn produces a more sensitive assay. An often-cited paper correctly rallies against this assumption and recommends the use of the highest affinity ligand available for experiments aiming to determine competitive inhibitor affinities. However, we have noted this advice being applied incorrectly to competition-based primary screens where the goal is optimum assay sensitivity, enabling a clear yes/no binding determination for even low affinity interactions. The published advice only applies to secondary, confirmatory assays intended for accurate affinity determination of primary screening hits. We demonstrate that using very high affinity ligands in competition-based primary screening can lead to reduced assay sensitivity and, ultimately, the discarding of potentially valuable active compounds. We build on techniques developed in our PyBindingCurve software for a mechanistic understanding of complex biological interaction systems, developing the "CLAffinity tool" for simulating competition experiments using protein, ligand, and inhibitor concentrations common to drug screening campaigns. CLAffinity reveals optimum labeled ligand affinity ranges based on assay parameters, rather than general rules to optimize assay sensitivity. We provide the open source CLAffinity software toolset to carry out assay simulations and a video summarizing key findings to aid in understanding, along with a simple lookup table allowing identification of optimal dynamic ranges for competition-based primary screens. The application of our freely available software and lookup tables will lead to the consistent creation of more performant competition-based primary screens identifying valuable hit compounds, particularly for difficult targets.
Sujet(s)
Protéines , Logiciel , Ligands , Liaison aux protéines , Protéines/composition chimiqueRÉSUMÉ
Background: Lower survival rates for many cancer types correlate with changes in nuclear size/scaling in a tumor-type/tissue-specific manner. Hypothesizing that such changes might confer an advantage to tumor cells, we aimed at the identification of commercially available compounds to guide further mechanistic studies. We therefore screened for Food and Drug Administration (FDA)/European Medicines Agency (EMA)-approved compounds that reverse the direction of characteristic tumor nuclear size changes in PC3, HCT116, and H1299 cell lines reflecting, respectively, prostate adenocarcinoma, colonic adenocarcinoma, and small-cell squamous lung cancer. Results: We found distinct, largely nonoverlapping sets of compounds that rectify nuclear size changes for each tumor cell line. Several classes of compounds including, e.g., serotonin uptake inhibitors, cyclo-oxygenase inhibitors, ß-adrenergic receptor agonists, and Na+/K+ ATPase inhibitors, displayed coherent nuclear size phenotypes focused on a particular cell line or across cell lines and treatment conditions. Several compounds from classes far afield from current chemotherapy regimens were also identified. Seven nuclear size-rectifying compounds selected for further investigation all inhibited cell migration and/or invasion. Conclusions: Our study provides (a) proof of concept that nuclear size might be a valuable target to reduce cell migration/invasion in cancer treatment and (b) the most thorough collection of tool compounds to date reversing nuclear size changes specific to individual cancer-type cell lines. Although these compounds still need to be tested in primary cancer cells, the cell line-specific nuclear size and migration/invasion responses to particular drug classes suggest that cancer type-specific nuclear size rectifiers may help reduce metastatic spread.
Sujet(s)
Adénocarcinome , Tumeurs de la prostate , Lignée cellulaire tumorale , Mouvement cellulaire , Humains , Mâle , Invasion tumorale/génétique , Invasion tumorale/prévention et contrôle , Tumeurs de la prostate/traitement médicamenteuxRÉSUMÉ
Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.
Sujet(s)
Diabète de type 2 , Transporteur de glucose de type 4 , Humains , Insuline , Microscopie de fluorescence , Transport des protéinesRÉSUMÉ
Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing Pseudomonas stutzeri have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of P. stutzeri biofilms, in combination with consortia-based approaches, may offer advantages for these processes. Understanding the interspecific interaction within biofilm co-cultures or consortia provides a means for improvement of current technologies. However, the investigation of biofilm-based consortia has been limited. We present an adaptable and scalable method for the analysis of macroscopic interactions (colony morphology, inhibition, and invasion) between colony-forming bacterial strains using an automated printing method followed by analysis of the genes and metabolites involved in the interactions. Using Biofilm Interaction Mapping and Analysis (BIMA), these interactions were investigated between P. stutzeri strain RCH2, a denitrifier isolated from chromium (VI) contaminated soil, and 13 other species of pseudomonas isolated from non-contaminated soil. One interaction partner, Pseudomonas fluorescens N1B4 was selected for mutant fitness profiling of a DNA-barcoded mutant library; with this approach four genes of importance were identified and the effects on interactions were evaluated with deletion mutants and mass spectrometry based metabolomics.
RÉSUMÉ
The identification of modulators for proteins without assayable biochemical activity remains a challenge in chemical biology. The presented approach adapts a high-throughput fluorescence binding assay and functional chromatography, two protein-resin technologies, enabling the discovery and isolation of fluorescent natural product probes that target proteins independently of biochemical function. The resulting probes also suggest targetable pockets for lead discovery. Using human survivin as a model, we demonstrate this method with the discovery of members of the prodiginine family as fluorescent probes to the cancer target survivin.
RÉSUMÉ
RNA-protein interactions are central to all gene expression processes and contribute to a variety of human diseases. Therapeutic approaches targeting RNA-protein interactions have shown promising effects on some diseases that are previously regarded as 'incurable'. Here, we developed a fluorescent on-bead screening platform, RNA Pull-Down COnfocal NAnoscanning (RP-CONA), to identify RNA-protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7-1. Importantly, miR-7's primary target is an mRNA of α-synuclein, which contributes to the aetiology of Parkinson's disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-synuclein. This opens up new therapeutic avenues towards treatment of Parkinson's disease as well as provides a novel methodology to search for modulators of RNA-protein interaction.
Sujet(s)
Protéine-1 similaire à ELAV/antagonistes et inhibiteurs , microARN/antagonistes et inhibiteurs , Quercétine/pharmacologie , alpha-Synucléine/métabolisme , Évaluation préclinique de médicament/méthodes , Protéine-1 similaire à ELAV/métabolisme , Cellules HEK293 , Cellules HeLa , Humains , microARN/métabolisme , Microscopie confocale , ARN messager/métabolisme , alpha-Synucléine/génétiqueRÉSUMÉ
Understanding multicomponent binding interactions in protein-ligand, protein-protein, and competition systems is essential for fundamental biology and drug discovery. Hand-deriving equations quickly become unfeasible when the number of components is increased, and direct analytical solutions only exist to a certain complexity. To address this problem and allow easy access to simulation, plotting, and parameter fitting to complex systems at equilibrium, we present the Python package PyBindingCurve. We apply this software to explore homodimer and heterodimer formations culminating in the discovery that under certain conditions, homodimers are easier to break with an inhibitor than heterodimers and may also be more readily depleted. This is a potentially valuable and overlooked phenomenon of great importance to drug discovery. PyBindingCurve may be expanded to operate on any equilibrium binding system and allows definition of custom systems using a simple syntax. PyBindingCurve is available under the MIT license at https://github.com/stevenshave/pybindingcurve as the Python source code accompanied by examples and as an easily installable package within the Python Package Index.
Sujet(s)
Protéines , Logiciel , Simulation numériqueRÉSUMÉ
Millions of people are affected by hearing loss. Hearing loss is frequently caused by noise or aging and often associated with loss of pericytes. Pericytes populate the small vessels in the adult cochlea. However, their role in different types of hearing loss is largely unknown. Using an inducible and conditional pericyte depletion mouse model and noise-exposed mouse model, we show that loss of pericytes leads to marked changes in vascular structure, in turn leading to vascular degeneration and hearing loss. In vitro, using advanced tissue explants from pericyte fluorescence reporter models combined with exogenous donor pericytes, we show that pericytes, signaled by VEGF isoform A165 (VEGFA165), vigorously drive new vessel growth in both adult and neonatal mouse inner ear tissue. In vivo, the delivery of an adeno-associated virus serotype 1-mediated (AAV1-mediated) VEGFA165 viral vector to pericyte-depleted or noise-exposed animals prevented and regenerated lost pericytes, improved blood supply, and attenuated hearing loss. These studies provide the first clear-cut evidence that pericytes are critical for vascular regeneration, vascular stability, and hearing in adults. The restoration of vascular function in the damaged cochlea, including in noise-exposed animals, suggests that VEGFA165 gene therapy could be a new strategy for ameliorating vascular associated hearing disorders.
Sujet(s)
Cochlée/vascularisation , Surdité due au bruit/physiopathologie , Néovascularisation physiologique/génétique , Péricytes/anatomopathologie , Facteur de croissance endothéliale vasculaire de type A/génétique , Animaux , Oreille interne/vascularisation , Thérapie génétique , Surdité due au bruit/thérapie , Techniques in vitro , Souris , Souris transgéniquesRÉSUMÉ
Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.
Sujet(s)
Hippocampe/métabolisme , Protéomique/méthodes , Animaux , Femelle , Cytométrie en flux , Hippocampe/ultrastructure , Humains , Métabolisme lipidique/physiologie , Mâle , Microscopie électronique à transmission , Système nerveux/métabolisme , Système nerveux/ultrastructure , Névroglie/métabolisme , Agents neuromédiateurs/métabolisme , Caractères sexuels , Transduction du signal/physiologieRÉSUMÉ
Quantitative microdialysis is a traditional biophysical affinity determination technique. In the development of the detailed experimental protocol presented, we used commercially available equipment, rapid equilibrium dialysis (RED) devices (ThermoFisher Scientific), which means that it is open to most laboratories. The target protein and test compound are incubated in a chamber partitioned to allow only small molecules to transition to a larger reservoir chamber, then reversed-phase high performance liquid chromatography (RP-HPLC) or liquid chromatography-mass spectrometry (LC-MS) is used to determine the abundance of compound in each chamber. A higher compound concentration measured in the chamber that contains the target protein indicates binding. As a novel, and differentiating contribution, we present a protocol for mathematical analysis of experimental data. We provide the equations and the software to yield dissociation constants for the test compound-target protein complex up to 0.5 mM KD, and we quantitatively discuss the limitations of affinities in relation to measured compound concentrations.
RÉSUMÉ
Cryo-electron tomography maps often exhibit considerable noise and anisotropic resolution, due to the low-dose requirements and the missing wedge in Fourier space. These spurious features are visually unappealing and, more importantly, prevent an automated segmentation of geometric shapes, requiring a subjective and labor-intensive manual tracing. We developed a novel computational strategy for objectively denoising and correcting missing-wedge artifacts in homogeneous specimen areas of tomograms, where it is assumed that a template repeats itself across the volume under consideration, as happens in the case of filaments. In our deconvolution approach, we use a template and a map of corresponding template locations, allowing us to compensate for the information lost in the missing wedge. We applied the method to tomograms of actin-filament bundles of inner-ear stereocilia, which are critical for the senses of hearing and balance. In addition, we demonstrate that our method can be used for cell membrane detection.
Sujet(s)
Algorithmes , Artéfacts , Tomographie en microscopie électronique , Traitement d'image par ordinateurRÉSUMÉ
Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.
Sujet(s)
Transformation cellulaire néoplasique/anatomopathologie , Tumeurs colorectales/métabolisme , Tumeurs colorectales/anatomopathologie , Récepteurs ErbB/métabolisme , Mutation/génétique , Cartes d'interactions protéiques , Protéines proto-oncogènes p21(ras)/génétique , Lignée cellulaire tumorale , Humains , Phosphorylation , Pronostic , Analyse de survie , Protéine Bad/métabolismeRÉSUMÉ
Electron cryo-tomography allows for high-resolution imaging of stereocilia in their native state. Because their actin filaments have a higher degree of order, we imaged stereocilia from mice lacking the actin crosslinker plastin 1 (PLS1). We found that while stereocilia actin filaments run 13 nm apart in parallel for long distances, there were gaps of significant size that were stochastically distributed throughout the actin core. Actin crosslinkers were distributed through the stereocilium, but did not occupy all possible binding sites. At stereocilia tips, protein density extended beyond actin filaments, especially on the side of the tip where a tip link is expected to anchor. Along the shaft, repeating density was observed that corresponds to actin-to-membrane connectors. In the taper region, most actin filaments terminated near the plasma membrane. The remaining filaments twisted together to make a tighter bundle than was present in the shaft region; the spacing between them decreased from 13 nm to 9 nm, and the apparent filament diameter decreased from 6.4 to 4.8 nm. Our models illustrate detailed features of distinct structural domains that are present within the stereocilium.